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United States Patent |
5,041,424
|
Saulnier
,   et al.
|
August 20, 1991
|
Epipodophyllotoxin glucoside 4'-phosphate derivatives
Abstract
Phosphate derivatives of 4'-demethylepipodophyllotoxin glucosides are novel
antitumor agents and the salts thereof offer the pharmaceutical advantage
of high water solubility.
Inventors:
|
Saulnier; Mark G. (Middletown, CT);
Senter; Peter D. (Seattle, WA);
Kadow; John F. (Meriden, CT)
|
Assignee:
|
Bristol-Myers Company (New York, NY)
|
Appl. No.:
|
450718 |
Filed:
|
December 14, 1989 |
Current U.S. Class: |
514/27; 514/33; 536/17.1; 536/18.1 |
Intern'l Class: |
A61K 031/70; C07H 015/24 |
Field of Search: |
536/17.1,18.1
514/27,33,35
|
References Cited
U.S. Patent Documents
3408441 | Oct., 1968 | Von Wartburg et al. | 536/18.
|
3524844 | Aug., 1970 | Keller-Juslen et al. | 536/18.
|
4564675 | Jan., 1986 | Kurabayashi et al. | 536/18.
|
Foreign Patent Documents |
63-192793 | Aug., 1988 | JP.
| |
Other References
Cancer Chemotherapy Reports, Part I (1975) 59:233-242.
|
Primary Examiner: Griffin; Ronald W.
Assistant Examiner: Carson; Nancy S.
Attorney, Agent or Firm: Yang; Mollie M.
Parent Case Text
This application is a divisional of U.S. patent application Ser. No.
199,731 filed on May 27, 1988 now U.S. Pat. No. 4,904,768 which is a
continuation-in-part of U.S. patent application, Ser. No. 081,493, filed
on Aug. 4, 1987, now abandoned, in the U.S. Patent and Trademark Office.
Claims
What is claimed is:
1. A method for inhibiting mammalian tumor said tumor being sensitive to
etoposide or teniposide which comprises administering to a mammal in need
thereof an antitumour effective amount of a compound of the formula
##STR10##
wherein R.sup.1 is methyl or 2-thienyl, and R.sup.6 is H; or a
pharmaceutically acceptable salt thereof.
2. A method according to claim 1 wherein said compound is etoposide
4'-phosphate disodium salt.
Description
BACKGROUND OF THE INVENTION
I. Field of the Invention
The present invention relates to 4'-phosphate derivatives of
epipodophyllotoxin glucosides, to their antitumor use, and to
pharmaceutical compositions containing these new agents.
II. Description of the Prior Art
Etoposide (VP-16, I) and teniposide (VM-26, II) are clinically useful
anticancer agents derived from the naturally occurring lignan,
podophyllotoxin (III): the class of compounds including etoposide and
teniposide is sometimes referred to as 4'-demethylepipodophyllotoxin
glucosides. Etoposide and teniposide are active in the treatment of a
variety of cancers including testicular, small cell lung, ovarian, breast,
thyroid, bladder, brain, non-lymphocytic leukemia, and Hodgkin's disease.
Compounds I and II, and the method for producing them are disclosed in U.S.
Pat. Nos. 3,408,441 to Wartburg et al and 3,524,844 to Keller-Juslen et
al. The compounds disclosed therein, in particular etoposide and
teniposide, serve as starting material for our preparation of
epipodophyllotoxin glucoside 4'-phosphate derivatives of the present
invention.
##STR1##
Phosphorylation of therapeutic agents containing a hydroxyl group has been
used as a means for drug latentiation; the phosphorylated derivatives may
then be cleaved in vivo by a phosphatase to liberate the active parent
molecule. A brief discussion of phosphates as potential prodrugs is
included in the review article entitled "Rational for Design of
Biologically Reversible Drug Derivatives: Prodrugs" (Sinkula and
Yalkowsky, J. Pharm Sci., 1975, 64:181-210 at 189-191). Examples of
phosphates of known antitumor agents include camptothecin (Japan Kokai No.
21-95,394 and 21-95,393, Derwent Abst. No. 87-281016 and 87-281015,
respectively) and daurorubicin (U.S. Pat. No. 4,185,111).
Podophyllotoxin phosphate disodium salt IV was prepared by Seligman et al.
However, the phosphate was not hydrolyzed by prostatic acid phosphatase
and did not show reduced toxicity over the parent podophyllotoxin (Cancer
Chemotherapy Reports Part I, 1975, 59:233-242).
##STR2##
The present invention provides phosphate esters of
4'-demethylepipodophyllotoxin glucosides which are active antitumor
agents. In particular, the dihydrogen phosphate of
4'-demethylepipodophyllotoxin glucosides and salts thereof are highly
water-soluble thus providing a superior pharmaceutical advantage over the
current therapeutic agents of this class, etoposide and teniposide, which
have minimal water solubility.
SUMMARY OF THE INVENTION
The present invention provides 4'-phosphate derivatives of
4'-demethylepipodophyllotoxin glucosides of general formula V, and
pharmaceutically acceptable salts thereof
##STR3##
wherein R.sup.6 is H and R.sup.1 is selected from the group consisting of
(C.sub.1-10)alkyl; (C.sub.2-10)alkenyl; (C.sub.5-6)cycloalkyl; 2-furyl;
2-thienyl; (C.sub.6-10)aryl; (C.sub.7-14)aralkyl; and
(C.sub.8-14)aralkenyl wherein each of the aromatic rings may be
unsubstituted or substituted with one or more groups selected from halo,
(C.sub.1-8)alkyl, (C.sub.1-8)alkoxy, hydroxy, nitro, and amino; or R.sup.1
and R.sup.6 are each (C.sub.1-8)alkyl; or R.sup.1 and R.sup.6 and the
carbon to which they are attached join to form a (C.sub.5-6) cycloalkyl
group; X is oxygen or sulfur; R.sup.7 and R.sup.8 are independently
selected from the group consisting of H, (C.sub.1-5) alkyl, A-substituted
(C.sub.1-5)alkyl, (C.sub.3-6)cycloalkyl, A-substituted
(C.sub.3-6)cycloalkyl, (C.sub.6-10)aryl, A-substituted aryl,
alkyl-substituted aryl, (C.sub.7-14)aralkyl, A-substituted aralkyl, and
alkyl-substituted aralkyl; wherein said A-substituents are one or more
groups selected from hydroxy, alkoxy, alkanoyloxy, cyano, amino,
alkylamino, dialkylamino, carboxy, alkylthio, mercapto, mercaptothio,
nitropyridyl disulfide, alkanoylamino, alkanoyl, carbamoyl, nitro, and
halo. The salts of compound V include both the monoanionic and the
dianionic salts. The cation may be a metal ion such as one from the alkali
metal or alkaline earth metal groups or other common metal ions; or an
organic nitrogen-containing group such as ammonium, mono-, di-, or
trialkylammonium, or pyridinium. The cation is preferably selected from
the group consisting of sodium, potassium, lithium, cesium, magnesium,
calcium, aluminum, ammonium and mono-,di-, and trialkylammonium. A
preferred embodiment provides compounds of formula V wherein R.sup.7 and
R.sup.8 are both H, and pharmaceutically acceptable salts thereof. A most
preferred embodiment provides etoposide 4'-dihydrogen phosphate and
thiophosphate, and their respective disodium salts VIa and VIb. A further
preferred embodiment provides
##STR4##
compounds of formula V wherein R.sup.7 and R.sup.8 are the same and are
selected from the group consisting of 2,2,2-trihaloethyl, 2-cyanoethyl,
(C.sub.1-5)alkyl, phenyl, and phenylalkyl, wherein the phenyl ring is
optionally substituted with alkyl, halogen, or nitro.
A further aspect of this invention provides antitumor phosphoroamidate
derivatives of formula VII and pharmaceutically acceptable salts thereof,
##STR5##
wherein R.sup.1, R.sup.6, and X are as previously defined; Y is Cl, OH, or
NR.sup.4 R.sup.5 ; R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are each
independently selected from the group consisting of H, (C.sub.1-5) alkyl,
(C.sub.2-5) alkenyl, (C.sub.3-6) cycloalkyl, A-substituted (C.sub.1-5)
alkyl, A-substituted (C.sub.2-5) alkenyl, A-substituted (C.sub.3-6)
cycloalkyl; or R.sup.2, R.sup.3, and the nitrogen to which they are
attached together represent a 3- to 6-membered ring; or R.sup.4, R.sup.5,
and the nitrogen to which they are attached together represent a 3- to
6-membered ring; wherein said A-substituents are as previously defined.
Another aspect of the present invention provides dichlorophosphate
intermediates of formula VIII wherein R.sup.1, R.sup.6 and X are as
previously defined; these agents are useful in the preparation of
compounds of formula V.
##STR6##
Yet a further aspect of the present invention provides a process for
preparing a compound of formula V wherein R.sup.7 and R.sup.8 are both H
and its pharmaceutically acceptable salts, which comprises the steps of
(a) converting a compound of formula IX
##STR7##
into a compound of formula X wherein R.sup.1, R.sup.6, and X are as
previously defined and G is a phosphate protecting group; (b) removing the
phosphate protecting group; and (c)
##STR8##
optionally converting the product of step (b) to a pharmaceutically
acceptable salt. Phosphate protecting groups include, but are not limited
to, those within the definition of R.sup.7 given above except H.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, unless otherwise specified, the term "alkyl" means straight
or branched carbon chains; "halo" includes bromo, chloro, fluoro, and
iodo; "etopofos" is the compound etoposide 4'-phosphate disodium salt
[i.e. compound VIa].
The phenol group of 4'-demethylepipodophyllotoxin glucosides may be
phosphorylated with phosphorous oxychloride and thiophosphoryl chloride to
give the corresponding dichlorophosphate and dichlorothiophosphate,
respectively (formula VIII). The phosphorylation reaction is performed in
a suitable anhydrous organic solvent, for example acetonitrile, and
preferably in the presence of a tertiary amine base, for example
N,N-diisopropylethylamine. The course of the reaction may be monitored by
thin layer chromatography (TLC) by which the optimum reaction time may be
judged by the appearance of product or the disappearance of the starting
material, or both. In our experience, the reaction period may take from
about 4 hours to about 72 hours. The length of reaction time required
appears to be related to the quality of the phosphorous reagent used.
The 4'-dichlorophosphates of formula VIII are versatile intermediates which
may subsequently react with nucleophiles to provide a variety of phosphate
and thiophosphate derivatives. Thus the intermediates may be hydrolyzed to
provide the phosphates, and in the presence of a base the phosphate salts
are obtained. For example, VIII treated with an excess of aqueous sodium
bicarbonate solution provides the corresponding 4'-phosphate disodium and
4'-thiophosphate disodium salts; bicarbonates of other cations such as
potassium and ammonium may also be used to provide the respective salts.
The dichlorophosphate intermediate VIII may react with amines to afford
either the corresponding phosphorodiamidate or the
chlorophosphoromonoamidate. Examples of suitable amines include, but are
not limited to, ammonia, primary amines such as ethylamine,
chloroethylamine, allylamine, dimethylaminopropylamine, hydroxyethylamine,
cyclohexylamine, and aminocyclohexanol; and secondary amines such as
diethylamine, piperidine, ethylmethylamine, methylaminoethanol,
ethylbutylamine, and the like. The amount of the amine used relative to
that of the epidpodophyllotoxin dichlorophosphate may be adjusted so as to
favor one or the other reaction product. For example, when a large excess
of the amine relative to the epipodophyllotoxin is used, the symmetrical
phosphorodiamidate is obtained, i.e. compounds of formula VII wherein Y is
the same as NR.sup.2 R.sup.3 ; the chlorophosphoromonoamidate, i.e.
compounds of formula VII wherein Y is Cl, may be prepared when a more
controlled amount of the amine is used. The chlorophosphoromonoamidate may
be hydrolyzed to provide compounds of formula VII wherein Y is OH or its
salts, or it may react further with a second amine to provide the
unsymmetrical phosphorodiamidate, i.e. compounds of formula VII wherein Y
is NR.sup.4 R.sup.5 and is different from NR.sup.2 R.sup.3.
The above-described procedure is illustrated in the following reaction
scheme.
##STR9##
Phosphate triesters are compounds of formula V wherein R.sup.7 and R.sup.8
are not H, and they may be prepared by treating a
4'-demethylepipodophyllotoxin glucoside with a halophosphate diester,
[i.e. Hal-P(X)(OR.sup.7)(OR.sup.8)]. It has been found that this reaction
is most efficiently performed in acetonitrile in the presence of an
organic trialkylamine base; the preferred base is diisopropylethylamine.
At least one equivalent of the halophosphate and the amine base is used,
but both reagents are preferably employed in molar equivalents in slight
excess relative to that of the epipodophyllotoxin glucoside reactant. The
reaction may be carried out at any temperature conducive to product
formation; however, slightly elevated temperatures, e.g.
30.degree.-40.degree. C. appear to facilitate the reaction which may take
up to several days to go to completion. Symmetrical halophosphate diesters
[i.e. R.sup.7 .dbd.R.sup.8 ] may be conventionally prepared from the
alcohol and e.g. phosphoryl chloride, and unsymmetrical ones [i.e. R.sup.7
.noteq.R.sup.8 ] may be prepared from the alcohol and dihalophosphate
ester. It is also possible to prepare phosphate triesters by other routes,
for example by first converting the phenol into a phosphite ester, e.g. by
reacting with a reagent such as (PhCH.sub.2 O).sub.2 PN(i-pr).sub.2, and
subsequently oxidizing the phosphate to the phosphate ester using e.g.
m-chloro perbenzoic acid.
Phosphate triesters may additionally serve as intermediates in the
preparation of compounds of formula V and salts thereof. Thus, for
example, the dihydroxy phosphate ( V, R.sup.7 .dbd.R.sup.8 .dbd.H) is
obtained when the diphenyl ester (V, R.sup.7 .dbd.R.sup.8 .dbd.phenyl) is
subjected to catalytic hydrogenation. Other suitable phosphate protecting
groups include but are not limited to, 2,2,2-trichloroethyl, benzyl,
cyanoethyl, p-nitro substituted phenyl, benzyl, phenethyl, and
p-bromophenyl. The dihydroxy phosphate (V, R.sup.7 .dbd.R.sup.8 .dbd.H)
are converted to base salts by reacting with the appropriate base, e.g.
sodium bicarbonate, ammonium bicarbonate or organic amines. Alternatively,
the salts may also be generated by eluting the dihydroxy phosphate through
a column of an exchange resin containing the desired cation.
Although the present invention utilizes phosphorous oxychloride,
halophosphate diesters, and their respective sulfur analogs as the
phosphorylating reagent, it is to be understood that other phosphorous
reagents capable of phosphorylating phenols may also be used, and
appropriate reaction conditions and medium may be chosen according to the
phosphorylating agent selected. The review article entitled "Current
Methods of Phosphorylation of Biological Molecules" (Synthesis, 1977,
737-52) contains further examples of phosphorylating agents and is hereby
incorporated by reference.
BIOLOGICAL PROPERTIES
Representative compounds of the present invention were evaluated for
antitumor activity against transplantable murine P388 leukemia. In all
experiments female CDF.sub.1 mice implanted with a tumor inoculum of
10.sup.6 ascites cells of P388 murine leukemia were used. In experiments
using etoposide 4'-phosphate, its disodium salt, and etoposide
4'-thiophosphate disodium salt, tumor implantation and drug treatment were
both via the iv route. In all other experiments tumor implant and drug
treatment were via the ip route. In all cases, however, the positive
control, etoposide, was administered ip. The experiments lasted 28 to 46
days at the end of which time the number of surviviors was noted.
Antitumor activity is expressed as % T/C which is the ratio of the median
survival time (MST) of drug-treated group to the MST of saline-treated
control group. A compound having % T/C value of 125 or greater is
generally considered to have significant antitumor activity in the P388
test. Table I presents the results of the above-described evaluation; the
maximum % T/C values and doses giving that effect are reported.
TABLE I
______________________________________
Antitumor Activity Against Murine P388 Leukemia.
Dose*
Compound of
(mg/kg/inj)
Route MST(d) % T/C
______________________________________
TUMOR CELLS IMPLANTED INTRAVENOUSLY
Example 1 140 IV 29.0 363
(Etoposide)
50 IP 20.5 256
Example 4 200 IP 18.0 225
(Etoposide)
100 IP 21.5 269
Example 8 125 IV 24.5 306
(Etoposide)
100 IP 29.5 369
TUMOR CELLS IMPLANTED INTRAPERITONEALLY
Example 2 240 IP 16.5 165
(Etoposide)
60 IP 25.0 250
Example 3 200 IP 15.5 155
(Etoposide)
100 IP 27.0 270
Example 7 240 IP 25.0 250
(Etoposide)
100 IP 26.0 260
Example 9 150 IP 19.5 217
(Etoposide)
100 IP 24.0 267
______________________________________
*Drugs were administered on day 5 and 8 unless otherwise specified (day 1
being the day of tumor implantation).
The antitumor compounds of the present invention have been demonstrated to
be active against transplanted tumors in experimental animals.
Specifically, the compound represented by formula VIa ("etopofos") shows
significantly higher antitumor activity than etoposide in the P388 test.
This selective agent represents a highly water soluble pro-drug of
etoposide which has reduced antitumor activity in-vitro and is rapidly
cleaved by alkaline phosphatase resulting in the release of etoposide. The
etoposide that is released exhibits identical cytotoxicity to the parent
drug.
Accordingly, the present invention provides a method for inhibiting
mammalian tumors which comprises administering an effective
tumor-inhibiting dose of an antitumor compound of formula V or VII to a
tumor bearing host. For this purpose, the drug may be administered by
conventional routes including, but not limited to, intravenous,
intramuscular, intratumoral, intraarterial, intralymphatic, and oral.
A further aspect of the present invention provides a pharmaceutical
composition which comprises a compound of formula V or VII and a
pharmaceutically acceptable carrier. The antitumor composition may be made
up of any pharmaceutical form appropriate for the desired route of
administration. Examples of such compositions include solid compositions
for oral administration such as tablets, capsules, pills, powders and
granules, liquid compositions for oral administration such as solutions,
suspensions, syrups or elixirs and preparations for parenteral
administration such as sterile solutions, suspensions or emulsions. They
may also be manufactured in the form of sterile solid compositions which
can be dissolved in sterile water, physiological saline or some other
sterile injectable medium immediately before use.
Optimal dosages and regimens for a given mammalian host can be readily
ascertained by those skilled in the art. It will, of course, be
appreciated that the actual dose used will vary according to the
particular composition formulated, the particular compound used, the mode
of application and the particular site, host and disease being treated.
Many factors that modify the action of the drug will be taken into account
including age, weight, sex, diet, time of administration, route of
administration, rate of excretion, condition of the patient, drug
combinations, reaction sensitivities and severity of the disease.
The following examples are for illustrative purposes only and should not be
construed as limiting the scope of the invention which is defined solely
by the Claims appended to this application.
In the following examples, proton and carbon nuclear magnetic resonance
(NMR) spectra (using CDCl.sub.3 or D.sub.2 O as an internal reference) and
phosphorous NMR spectra (using 85% aqueous H.sub.3 PO.sub.4 as an external
reference) were recorded on a Bruker WM360 spectrometer. Infrared spectra
(IR) were determined on a Perkin-Elmer 1800 Fourier Transform Infrared
Spectrophotometer. "Flash chromatography" refers to the method described
by Still (Still, W. C.; Kahn, M.; Mitra, A.; J. Org. Chem., 1978 43, 2923)
and was carried out using E. Merck silica gel (230-400 mesh). Reverse
phase chromatography was carried out under a positive nitrogen pressure
using C.sub.18 (Octadecylsilane) bonded to silica gel (40 - .mu.m
diameter, J. T. Baker supplier).
EXAMPLE 1
Etoposide 4'-Phosphate Disodium Salt (Compound VIa)
A magnetically stirred suspension of etoposide (2.30 g, 3.91 mmol) in dry
acetonitrile (210 ml) was warmed to give a nearly complete solution. The
solution was allowed to cool to room temperature, and
N,N-diisopropylethylamine (2.36 ml, 13.5 mmol) was added. The mixture was
then cooled to 0.degree. C. and POCl.sub.3 (666 mg, 4.34 mmol) was added
via syringe over 30 seconds. The mixture was allowed to slowly come to
room temperature over 2-3 hours and stirring continued at room temperature
for 63 hours. At the end of this period 20% by volume was removed and
treated with diethylamine as described in Example 2. The remainder was
treated with a solution of sodium bicarbonate (6.0 g, 71.4 mmol) in
deionized H.sub.2 O (110 ml), the mixture was stirred at room temperature
for 80 minutes, and then partitioned with saturated aqueous sodium
bicarbonate (20 ml) deionized H.sub.2 O (125 ml), and ethyl acetate (350
ml). The organic layer was further extracted with deionized H.sub.2 O
(1.times.50 ml) and the combined aqueous layers were washed with ethyl
acetate (250 ml) and then subjected to a vacuum of 0.5 mm at room
temperature for 1 hour to remove dissolved solvents. The aqueous portion
was then applied to a 4 cm diameter column containing 15 cm of
octadecylsilane bonded to silica gel which had been packed in methanol and
equilibrated with H.sub.2 O. After all of the aqueous portion was applied,
the column was eluted with H.sub.2 O (175 ml) to remove inorganic salts
and then 4:1 H.sub.2 O:CH.sub.3 OH eluted the product. Concentration of
the solvent at 0.5 torr provided 744 mg (36%) of the pure title compound
as a colorless solid. Alternatively lyophilization provides the pure title
compound as a very fluffy low density solid.
IR (KBr) 3426, 1775, 1593, 1505, 1486, 1337, 1239, 1191, 1122, 1078, 1034,
983, 927, 888, 876, 851, 840, 697, 684, 664, 547 cm.sup.-1.
360 MHz .sup.1 H NMR (D.sub.2 O) .delta. 6.93 (s,1H), 6.59 (s,1H), 6.27
(s,2H), 5.93 (d,2H), 5.09 (d,1H,J=2.8 Hz), 4.83 (q,1H,J=5.0 Hz), 4.68
(d,1H,J=7.9 Hz), 4.62 (d,.1H,J=5.7 Hz), 4.47-4.35 (m,2H), 4.24
(dd,1H,J=4.4 and 10.4 Hz), 3.64 (s,6H), 3.68-3.52 (m,3H), 3.44-3.30
(m,3H), 3.17-3.07 (m,1H), 1.31 (d,3H,J=5.0 Hz).
90 MHz .sup.13 C NMR (D.sub.2 O) .delta. 178.5, 151.8, 148.1, 146.1, 135.0,
132.6, 130.9, 127.4, 109.9, 109.5, 107.4, 101.3, 100.4, 99.6, 79.2, 73.7,
72.7, 72.2, 69.1, 67.1, 65.4, 55.6, 42.8, 40.3, 37.5, 18.8.
146 MHz .sup.31 P NMR (D.sub.2 O) .delta. 3.79.
Mass spectrum (FAB), m/e, 713 (M.sup.+ +H). C.sub.29 H.sub.31 Na.sub.2
O.sub.16 P requires M.sup.+, 712.
Anal. Calcd. for C.sub.29 H.sub.31 Na.sub.2 O.sub.16 P: C,48.89; H,4.39;
Na,6.45. Found*: C,48.72; H,4.56; Na,6.56.
*Adjusted for 8.16% H.sub.2 O determined by Karl Fischer analysis.
EXAMPLE 2
Etoposide 4'-(Bis-[N,N-diethyl]phosphonamide) (VII, X.dbd.O, R.sup.1
.dbd.methyl, R.sup.6 .dbd.H, Y.dbd.N(Et).sub.2, R.sup.2 .dbd.R.sup.3
.dbd.Et)
As indicated in Example 1, 20% by volume of the reaction product mixture of
etoposide and POCl.sub.3 was added to diethylamine (4 mL) and stirred at
room temperature for 3 hours. The solvent was evaporated in vacuo and the
light orange residue purified by flash chromatography on silica gel.
Elution with 4% methanol in methylene chloride provided 271.3 mg (46.9%)
of the pure title compound as a light yellow solid.
IR (KBr) 3408, 2974, 2936, 2877, 1774, 1598, 1508, 1486, 1467, 1421, 1383,
1339, 1234, 1191, 1162, 1130, 1098, 1079, 1037, 902, 858, 795, 713, 700,
544 cm.sup.-1.
360 MHz .sup.1 H NMR (CDCl.sub.3) .delta. 6.79, (s,1H), 6.50 (s,1H), 6.20
(s,2H), 5.96 (ABq,2H), 4.87 (d,1H,J=3.2 Hz), 4.71 (q,1H,J=5.1Hz), 4.61
(d,1H,J=7.6 Hz), 4.57 (d,1H,J=5.2 Hz), 4.39 (dd,1H,J=9.1 and 10.2 Hz),
4.22-4.13 (m,2H), 3.74 (m,1H), 3.65 (s,6H), 3.55 (m,1H), 3.40 (m,1H),
3.32-3.10 (m,11H), 2.94-2.83 (m,1H), 1.37 (d,3H,J=5.1Hz), 1.10 (m,12H).
146 MHz .sup.31 P NMR (CDCl.sub.3) .delta. 16.49.
Mass spectrum (FAB), m/e, 779 (M.sup.+ +H), 573 (M.sup.+ - sugar).
C.sub.37 H.sub.51 N.sub.2 O.sub.14 P requires M.sup.+, 778.
EXAMPLE 3
Etoposide 4'-(N,N-[2-chloroethyl]phosphoryl chloride) (VII, R.sup.1
.dbd.methyl, R.sup.6 .dbd.H, X.dbd.O, Y.dbd.Cl, R.sup.2 .dbd.R.sup.3
.dbd.CH.sub.2 CH.sub.2 Cl)
A magnetically stirred suspension of etoposide (2.00 g, 3.40 mmol) in dry
acetonitrile (220 mL), was warmed to give a nearly complete solution. The
mixture was cooled to room temperature and treated with
N,N-diisopropylethylamine (2.05 mL, 11.8 mmol). The mixture was then
cooled to 0.degree. C. under N.sub.2 and phosphorous oxychloride (624 mg,
4.07 mmol) added by syringe over 30 seconds. The mixture was magnetically
stirred at 0.degree. C. for 2.5 hours and then at room temperature for an
additional 1.5 hours. Bis-(2-chlorethylamine) hydrochloride (1.82 g, 10.2
mmol) was then rapidly added followed immediately by additional
N,N-diisopropylethylamine (2.10 mL, 12.0 mmol). The mixture was stirred at
room temperature for 85 minutes, concentrated in vacuo to a volume of
about 5 mL, and dissolved in ethyl acetate (400 mL) and methanol (5 mL).
The resulting solution was washed with pH 5 buffer (2.times.200 mL), water
(150 mL), and brine (150 mL) and dried over Na.sub.2 SO.sub.4 /MgSO.sub.4.
Evaporation of the solvent gave a yellow orange solid which was purified
by flash chromatography on silica gel with 3-4% methanol in methylene
chloride to provide 1.25 g (45.4%) of the pure title compound as a
colorless solid.
360 MHz .sup.1 H NMR (CDCl.sub.3) .delta. 6.82 (s,1H), 6.52 (s,1H), 6.27
(s,2H), 5.99 (d,2H), 4.90 (d,1H,J=3.4 Hz), 4.73 (q,1H,J=5.0 Hz), 4.65-4.60
(m,2H), 4.41 (m,1H), 4.25-4.15 (m,2H), 3.75-3.65 (m,5H), 3.72 (s,6H),
3.60-3.23 (m,9H), 2.91-2.80 (m,1H), 1.38 (d,3H,J=5.0 Hz).
146 MHz .sup.31 P NMR (CDCl.sub.3) .delta. 11.16 and 10.96 (two peaks due
to chiral phosphorous).
Mass spectrum (FAB), m/e, 812, 810, 808. C.sub.33 H.sub.39 Cl.sub.3
NO.sub.14 P requires M.sup.+ (.sup.35 Cl) 809.
EXAMPLE 4
Etoposide 4'-Thiophosphate Disodium Salt (Compound VIb)
A magnetically stirred suspension of etoposide (2.04 g, 3.47 mmol) in dry
acetonitrile (175 mL) was warmed to give a nearly complete solution. The
solution was allowed to cool to room temperature and
N,N-diisopropylethylamine (2.00 mL, 11.5 mmol) was then added thereto. The
mixture was then cooled to 0.degree. C. and thiophosphoryl chloride (0.720
g, 4.17 mmol) was added via syringe over a 30 second period. The mixture
was allowed to slowly warm to room temperature over 2-3 hours and stirring
continued at room temperature for 16 hours. The mixture was then warmed to
30.degree.-35.degree. C. and kept at that temperature for an additional 4
hours. A major new spot of higher Rf than etoposide was observed by TLC
(5% CH.sub.3 OH in CH.sub.2 Cl.sub.2). The reaction mixture was treated
with solid sodium bicarbonate (7.4 g) and then deionized H.sub.2 O (100
mL) was added. The mixture was stirred at 28.degree.-25.degree. C. for 1.5
hours and at room temperature for 1.5 hours. The mixture was partitioned
with deionized H.sub.2 O (200 mL), saturated aqueous sodium bicarbonate
(30 mL) and ethyl acetate (300 mL). Further workup and reverse phase
chromatography was performed according to the procedure delineated in
Example 1 to provide 1.03 g (40.8%) of the pure title compound as a
colorless solid.
360 MHz .sup.1 H NMR (D.sub.2 O) .delta. 6.93 (s,1H), 6.60 (s,1H), 6.27
(s,2H), 5.93 (d,2H), 5.09, (d,1H,J=2.8 Hz), 4.83 (q,1H,J=5.0 Hz), 4.68
(d,1H,J=7.8 Hz), 4.63 (d,1H,J=5.7 Hz), 4.47-4.35 (m,2H), 4.24 (dd,1H,J=4.3
and 10.5 Hz), 3.64 (s,6H), 3.67-3.52 (m,3H), 3.47-3.29 (m,3H), 3.17-3.07
(m,1H), 1.31 (d,3H,J=5.0 Hz).
Mass spectrum (FAB), m/e 728 (M.sup.+), 706 (M.sup.+ +H - Na). C.sub.29
H.sub.31 Na.sub.2 O.sub.15 PS requires M.sup.+, 728.
EXAMPLE 5
Etoposide
4'-[[N,N-bis(2-chloroethyl)amino]-[N-(3-hydroxypropyl)amino]]phosphate
(VII, X.dbd.O, R.sup.1 .dbd.methyl, R.sup.6 .dbd.H, R.sup.2 .dbd.R.sup.3
.dbd.2--chloroethyl, Y.dbd.--NH(CH.sub.2).sub.3 OH
A magnetically stirred solution of the compound of Example 3 (280 mg, 0.346
mmol) in CH.sub.2 Cl.sub.2 (3 ml) was treated with a solution of
3-amino-1-propanol (33.5 mg, 0.446 mmol) in CH.sub.2 Cl.sub.2 (1 ml).
After 5 minutes additional 3-amino-1-propanol (31.0 mg, 0.413 mmol) in
absolute methanol (0.5 ml) was added. The reaction mixture was purified by
direct application to 4 preparative TLC plates (1 mm, E. Merck silica gel)
which were developed using 5-8% CH.sub.3 OH in CH.sub.2 Cl.sub.2. Elution
of the desired product band using 5% CH.sub.3 OH in ethyl acetate followed
by evaporation in vacuo and then further drying at 0.1 torr provided 185
mg (63%) of the pure title compound as a colorless solid (mixture of
diastereomers at phosphorus).
360 MHz .sup.1 H NMR (CDCl.sub.3) .delta. 7.20 (br s, 1H), 6.80 (s, 1H),
6.50 and 6.48 (2s, 1H), 6.26 and 6.25 (2s, 2H), 5.97 (d, 2H), 4.88 (m,
1H), 4.73 (q, 1H), 4.64-4.57.(m, 2H), 4.40 (m, 1H), 4.21-4.13 (m, 2H),
3.71, 3.70 (2s, 6H), 3.71-3.06 (m, 18H), 2.90-2.80 (m, 1H), 1.37 (d, 3H).
Mass Spectrum (FAB), m/e, 849, 851 (M.sup.+ +H, .sup.35 Cl, .sup.37 Cl).
C.sub.36 H.sub.47 Cl.sub.2 N.sub.2 O.sub.15 P requires M.sup.+ 848
(.sup.35 Cl) and 850 (.sup.37 Cl).
EXAMPLE 6
Etoposide
4'-[[N,N-bis(2-chloroethyl)amino]-[N-[2-[(3-nitro-pyridyl-2-yl)disulfide]et
hyl]]amino]phosphate (VII. X.dbd.O, R.sup.1 .dbd.methyl, R.sup.6 .dbd.H,
R.sup.2 .dbd.R.sup.3 .dbd.2-chloroethyl, Y.dbd.NH(CH.sub.2).sub.2
--SS-(3-nitropyridyl-2-yl)
A mixture of the compound of Example 3 (248 mg, 0.306 mmol) and
2-(3-nitropyridyl)-1-(2-aminoethyl) disulfide hydrochloride (105 mg, 0.393
mmol) was treated with CH.sub.2 Cl.sub.2 (7 ml) followed by the addition
of diisopropylethylamine (100 .mu.l, 0.570 mmol) and dry methanol (0.5
ml). The resulting solution was stirred at room temperature for 1.5 hours
and then purified by direct application to four preparative TLC plates (1
mm, E. Merck silica gel) which were developed using 4-5% CH.sub.3 OH in
ethyl acetate. Elution of the desired product band using 5% CH.sub.3 OH in
ethyl acetate followed by evaporation in vacuo and then further drying at
0.1 torr provided 231.7 mg (75.3%) of the pure title compound as a
yellow-brown solid (mixture of diastereomers at phosphorous).
IR (KBr) 1774, 1598, 1584, 1559, 1509, 1486, 1456, 1421, 1397, 1342, 1236,
1160, 1128, 1096, 1038, 1004, 926, 857, 747, 699 cm.sup.-1.
360 MHz .sup.1 H NMR (CDCl.sub.3) .delta. 8.81 and 8.77 (2m, 1H), 8.48 (m,
1H), 7.33 (m, 1H), 6.81 (s, 1H), 6.51 and 6.50 (2s, 1H), 6.26 (br s, 2H),
5.97 (d, 2H), 4.89 (m, 1H), 4.73 (q, 1H), 4.65-4.52 (m, 3H), 4.41 (m, 1H),
4.24-4.14 (m, 2H), 3.71, 3.70 (2s, 6H), 3.71-2.85 (m, 19 H), 2.68 (br s,
1H, OH), 2.37 (br s, 1H, OH), 1.37 (d, 3H).
Mass Spectrum (FAB), m/e, 1005, 1007 (M.sup.+ +H, .sup.35 Cl, .sup.37 Cl).
C.sub.40 H.sub.47 Cl.sub.2 N.sub.4 O.sub.16 PS.sub.2 requires M.sup.+,
1004 (.sup.35 Cl) and 1006 (.sup.37 Cl).
EXAMPLE 7
Etoposide
4'-diphenyl phosphate (R.sup.1 .dbd.CH.sub.3, R.sup.6 .dbd.H, R.sup.7
.dbd.R.sup.8 .dbd.phenyl)
A magnetically stirred suspension of etoposide (10.50 g, 17.84 mmol, dried
over P.sub.2 O.sub.5 at 80.degree. C./0.5 torr) in dry acetonitrile (450
ml) was treated with diisopropylethylamine (4.20 ml, 24.1 mmol) and then
diphenyl chlorophosphate (2.00 ml, 9.65 mmol) was added neat via syringe.
The mixture was stirred under N.sub.2 for two hours at 50.degree. C. at
which point all of the etoposide had dissolved. Additional diphenyl
chlorophosphate (1.80 ml, 8.68 mmol) was added and the reaction mixture
was held at 45.degree. C. for 72 hours. After more of the amine base (0.75
ml, 4.3 mmol) and diphenyl chlorophosphate (0.80 ml, 3.86 mmol) were
added, the mixture was stirred at 40.degree.-45.degree. C. for 27 hours,
treated with more diphenyl chlorophosphate (0.40 ml, 1.93 mmol), and
maintained at 40.degree.-45.degree. C. for 22 hours. Isopropanol (20 ml)
was then added, the solvent was evaporated in vacuo. and the solid residue
was dissolved in CH.sub.2 Cl.sub.2 (500 ml), and partitioned with H.sub.2
O (400 ml). The aqueous layer was further extracted with CH.sub.2 Cl.sub.2
(100 ml) and the combined organic extracts were washed with brine (250 ml)
and dried (Na.sub.2 SO.sub.4 /MgSO.sub.4). Rotary evaporation followed by
flash chromatography on silica gel using 2-3% CH.sub.3 OH in CH.sub.2
Cl.sub.2 provided 12.50 g (85%) of the pure title compound as a colorless
solid.
FAB MS m/e (relative intensity) 820 (M+H).sup.+.
IR (KBr) 3460, 2925, 1775, 1601, 1490 cm.sup.-1.
.sup.1 H NMR (CDCl.sub.3) .delta. 7.28 (m,8H), 7.15 (m,2H), 6.78 (s,1H),
6.47 (s,1H), 5.95 (m,2H), 4.85 (d,J=3.5 Hz,1H), 4.71 (m,1H), 4.60 (d,J=7.6
Hz,1H), 4.56 (d,J=5.1Hz,1H), 4.38 (m,1H), 4.22-4.13 (m,2H), 3.72-3.60
(m,1H), 3.48 (s,6H), 3.54-3.28 (m,3H), 3.23 (dd,J=14.2,5.3 Hz,1H), 2.78
m,1H), 1.35 (d,J=5.1Hz,3H).
Anal. Calcd. for C.sub.41 H.sub.41 O.sub.16 P: C,60.00; H,5.04. Found:
C,60.20; H,5.16.
EXAMPLE 8
Etoposide 4'-phosphate (V; R.sup.1 .dbd.CH.sub.3 ; R.sup.6 .dbd.H, R.sup.7
.dbd.R.sup.8 .dbd.H)
Platinum oxide (0.198 g, 0.87 mmol) from a freshly opened bottle (Aldrich
Chemical Co.) was added to a soulution of etoposide 4'-diphenyl phosphate
(product of Example 7; 0.79 g, 0.962 mmol) in 95 mL of absolute ethanol.
The solution was hydrogenated on a Parr apparatus under 45-50 PSI for 4 h
at room temperature. The reaction mixture was filtered through a pad of
celite using ethanol as eluent. Concentration in vacuo and drying over
P.sub.2 O.sub.5 for 14 h in vacuo provided the desired product as a white
solid (0.627,94%):
FAB MS m/e (relative intensity) 669 (M+H).sup.+
IR (KBr) 3440, 2930, 1778, 1604, 1498 cm.sup.-1.
.sup.1 H NMR (DMSO-d.sub.6) .delta. 6.93 (s,1H), 6.46 (s,1H), 6.12 (s,2H),
5.94 (m,2H), 5.17 (bs,1H), 4.86 (d,J=3.93 Hz,1H), 4.64 (q,J=7.5,5.8
Hz,1H), 4.51-4.42 (m,2H), 4.20 (d,J=10.7 Hz,1H), 4.01 (dd,J=12.1,5.3
Hz,1H), 3.51 (s,6H), 3.51-2.75 (m,7H), 2.83 (m,1H), 1.16 (d,J=5.1Hz,3H).
.sup.13 C NMR (DMSO-d.sub.6) .delta. 174.5, 151.2, 151.1, 147.7, 146.2,
126.1, 132.3, 128.8, 109.8, 109.7, 107.9, 101.5, 101.2, 98.5, 80.0, 74.3,
72.7, 71.7, 67.6, 67.2, 65.7, 55.8, 43.0, 37.1, 20.2, 18.5.
Anal. Calcd. for C.sub.29 H.sub.33 O.sub.16 P. 0.85% H.sub.2 O: C,50.95; H,
5.11. Found: C,51.42; H,4.97.
EXAMPLE 9
Etoposide 4'-bis(2,2,2-trichlorethyl)phosphate (VIII; R.sup.6 .dbd.CH ,
R.sup.1 .dbd.H, R.sup.7 .dbd.R.sup.8 .dbd.CH.sub.2 CCl.sub.3)
The procedure described in Example 7 was repeated using
bis(2,2,2-trichloroethyl)chlorophosphate to provide the title compound in
100% yield as a colorless solid following flash chromatography on silica
gel.
IR (KBr) 1780, 1610, 1490, 1415, 1345, 1240, 1040, 960, 725 cm.sup.-1.
300 MHz .sup.1 H NMR (CDCl.sub.3) .delta. 6.81 (s,1H), 6.49 (s,1H), 6.27
(s,2H), 5.98 (dd,2H), 4.88 (d,1H,J=3.4 Hz), 4.82-4.70 (m,5H), 4.64
(d,1H,J=7.6 Hz), 4.61 (d,1H,J=5.3 Hz), 4.41 (dd,1H), 4.25-4.13 (m,2H),
3.75 (m,1H), 3.73 (s,6H), 3.56 (m,1H), 3.43 (dd,1H), 3.34-3.24 (m,3H),
2.91-2.82 (m,1H), 1.38 (d,3H,J=4.9 Hz).
mass spectrum (FAB), m/e=928.9848 (M.sup.+ +H). C.sub.33 H.sub.36 Cl.sub.6
O.sub.16 P requires 928.9872.
EXAMPLE 10
Etoposide 4'-phosphate disodium salt from etoposide 4'-phosphate
Method A
Commercial Dowex 50.times.8-100 cation exchange resin in the hydrogen form
(20 g, Aldrich Chemical Co.) was treated with excess 1N NaOH. The
resulting resin in Na+ form was packed into a 2 cm column and equilibrated
with water. Etoposide 4'-phosphate (product of Example 8, 1.25 g, 1.87
mmol) dissolved in 25 ml of deionized water was applied to the top of the
packed column and the column was eluted with water. Fractions containing
the title compound were pooled, filtered, and lyophilized to yield 1.15 g
of the title compound as a white and fluffy material.
Method B
To 2.90 g (4.34 mmol) of crude etoposide 4'-phosphate (product of Example
8) was added deionized water (50 ml) and sodium bicarbonate (3.00 g, 35.7
mmol). The mixture was stirred at room temperature for 30 minutes during
which time CO.sub.2 evolution ceased. The mixture was then chromatographed
as described in Example 1. Elution with deionized water (300 ml) and then
4:1 H.sub.2 O/CH.sub.3 OH provided 1.90 g (61%) of pure title compound as
a fluffy white solid following lyophilization.
EXAMPLE 11
The general procedure described in Example 2 is repeated with the exception
that the diethylamine used therein is replaced by the amines listed below
to provide the corresponding etoposide 4'-phosphorodiamidates.
______________________________________
Compound VII (X = O,
R.sup.1 = methyl, R.sup.6 = H,
Y = NR.sup.2 R.sup.3)
Amine R.sup.2 R.sup.3
______________________________________
propylamine H CH.sub.2 CH.sub.2 CH.sub.3
ethanolamine H CH.sub.2 CH.sub.2 OH
methoxyethylamine
H CH.sub.2 CH.sub.2 OCH.sub.3
N-acetylethylenediamine
H CH.sub.2 CHNC(O)CH.sub.3
2-methylallylamine
H CH.sub.2 CH(CH.sub.3).dbd.CH.sub.2
allylamine H CH.sub.2 CH.dbd.CH.sub.2
dimethylaminopropylamine
H (CH.sub.2)N(CH.sub.3).sub.2
N-methylethylenediamine
H CH.sub.2 CH.sub.2 NCH.sub.3
trifluoroethylamine
H CH.sub.2 CF.sub.3
2-aminoethanethiol
H CH.sub.2 CH.sub.2 SH
cyclohexylamine H cyclohexyl
2-amino-1-methoxypropane
H CH(CH.sub.3)CH.sub.2 OCH.sub.3
2-(ethylthio)-ethylamine
H CH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.3
chloroethylamine
H CH.sub.2 CH.sub.2 Cl
4-aminocyclohexanol
H
ethylmethylamine
CH.sub.3 CH.sub.2 CH.sub.3
ethylbutylamine CH.sub.2 CH.sub.3
(CH.sub.2).sub.3 CH.sub.3
methylaminoethanol
CH.sub.3 CH.sub.2 CH.sub.2 OH
bis(2-chloroethyl)amine
CH.sub.2 CH.sub.2 Cl
CH.sub.2 CH.sub.2 Cl
2-propylaminoethanol
CH.sub.2 CH.sub.2 CH.sub.3
CH.sub.2 CH.sub.2 OH
3-methylaminopropionitrile
CH.sub.3 CH.sub.2 CH.sub.2 CN
piperidine R.sup.2 + R.sup.3 =
--(CH.sub.2).sub.5 --
______________________________________
EXAMPLE 12
The general procedure described in Example 3 is repeated with the exception
that the bis(2-chloroethyl)amine used there is replaced by the amines
listed below to provide the corresponding etoposide chlorophoroamidates.
______________________________________
Compound VII (X = O,
R.sup.1 = methyl, R.sup.6 = H,
Y = Cl)
Amine R.sup.2 R.sup.3
______________________________________
propylamine H CH.sub.2 CH.sub.2 CH.sub.3
ethanolamine H CH.sub.2 CH.sub.2 OH
methoxyethylamine
H CH.sub.2 CH.sub.2 OCH.sub.3
N-acetylethylenediamine
H CH.sub.2 CHNC(O)CH.sub.3
2-methylallylamine
H CH.sub.2 CH(CH.sub.3).dbd.CH.sub.2
allylamine H CH.sub.2 CH.dbd.CH.sub.2
dimethylaminopropylamine
H (CH.sub.2)N(CH.sub.3).sub.2
N-methylethylenediamine
H CH.sub.2 CH.sub.2 NCH.sub.3
Trifluoroethylamine
H CH.sub.2 CF.sub.3
2-aminoethanethiol
H CH.sub.2 CH.sub.2 SH
cyclohexylamine H cyclohexyl
2-amino-1-methoxypropane
H CH(CH.sub.3)CH.sub.2 OCH.sub.3
2-(ethylthio)-ethylamine
H CH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.3
chloroethylamine
H CH.sub.2 CH.sub.2 Cl
4-aminocyclohexanol
H 4-OH cyclohexyl
ethylmethylamine
CH.sub.3 CH.sub.2 CH.sub.3
ethylbutylamine CH.sub.2 CH.sub.3
(CH.sub.2).sub.3 CH.sub.3
methylaminoethanol
CH.sub.3 CH.sub.2 CH.sub.2 OH
diethylamine CH.sub.2 CH.sub.3
CH.sub.2 CH.sub.3
2-propylaminoethanol
CH.sub.2 CH.sub.2 CH.sub.3
CH.sub.2 CH.sub.2 OH
3-methylaminopropionitrile
CH.sub. 3 CH.sub.2 CH.sub.2 CN
piperidine R.sup.2 + R.sup.3 =
--(CH.sub.2).sub.5 --
______________________________________
EXAMPLE 13
The general procedure in Example 5 is repeated with the exception that the
3-aminopropanol used therein is replaced by the following amines to
provide the corresponding unsymmetrical etoposide phosphorodiamidates.
______________________________________
Compound VII (X = O, - R.sup.1 = methyl, R.sup.6 = H,
Y = NR.sup.4 R.sup.5,
R.sup.2 = R.sup.3 = CH.sub.2 CH.sub.2 Cl)
Amine R.sup.4 R.sup.5
______________________________________
propylamine H CH.sub.2 CH.sub.2 CH.sub.3
ethanolamine H CH.sub.2 CH.sub.2 OH
methoxyethylamine
H CH.sub.2 CH.sub.2 OCH.sub.3
N-acetylethylenediamine
H CH.sub.2 CHNC(O)CH.sub.3
2-methylallylamine
H CH.sub.2 CH(CH.sub.3).dbd.CH.sub.2
allylamine H CH.sub.2 CH.dbd.CH.sub.2
dimethylaminopropylamine
H (CH.sub.2)N(CH.sub.3).sub.2
N-methylethylenediamine
H CH.sub.2 CH.sub.2 NCH.sub.3
trifluoroethylamine
H CH.sub.2 CF.sub.3
2-aminoethanethiol
H CH.sub.2 CH.sub.2 SH
cyclohexylamine H cyclohexyl
2-amino-1-methoxypropane
H CH(CH.sub.3)CH.sub.2 OCH.sub.3
2-(ethylthio)-ethylamine
H CH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.3
chloroethylamine
H CH.sub.2 CH.sub.2 Cl
4-aminocyclohexanol
H 4-OH cyclohexyl
ethylmethylamine
CH.sub.3 CH.sub.2 CH.sub.3
ethylbutylamine CH.sub.2 CH.sub.3
(CH.sub.2).sub.3 CH.sub.3
methylaminoethanol
CH.sub.3 CH.sub.2 CH.sub.2 OH
bis(2-chloroethyl)amine
CH.sub.2 CH.sub.2 Cl
CH.sub.2 CH.sub.2 Cl
2-propylaminoethanol
CH.sub.2 CH.sub.2 CH.sub.3
CH.sub.2 CH.sub.2 OH
3-methylaminopropionitrile
CH.sub.3 CH.sub.2 CH.sub.2 CN
piperidine R.sup.2 + R.sup.3 =
--(CH.sub.2).sub.5 --
______________________________________
EXAMPLE 14
The general procedure described in Example 7 is repeated with the exception
that the diphenyl chlorophosphate used therein is replaced with the
chlorophosphates listed below to provide the corresponding etoposide
4'-phosphate diesters (X.dbd.O, R.sup.1 .dbd.methyl, R.sup.6 .dbd.H,
R.sup.7 .dbd.R.sup.8 .dbd.R described below).
______________________________________
chlorophosphates [(RO).sub.2 P(O)Cl]
______________________________________
R = methyl
ethyl
benzyl
p-nitrobenzyl
p-nitrophenyl
p-bromobenzyl
p-nitrophenethyl
cyanoethyl
o-(t-butyl)phenyl
______________________________________
EXAMPLE 15
The general procedures described in Examples 1 to 16 are repeated with the
exception that the etoposide starting materials used therein are replaced
with the corresponding teniposide compounds to provide the corresponding
teniposide products.
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